The MicroRNAs Expression In The Durg Resistance Hepatocellular Carcinoma, Cell Line Induced By Doxorubicin And The Role Of MiR-1246in Drug Resistance Hepatocellular Carcinoma Cell Line

Chapter I Establishment of drug resistant cell line SMMC-7721/ADM and miRNAs expression profiling among SMMC-7721, LO2and SMMC-7721/ADMObjectiveTo investigate the miRNAs differentially expression in LO2, SMMC-7721and drug resistant cell line SMMC-7721/ADM.Methods(1) MTT was used to determine the IC50of Doxorubcin on SMMC-7721, established SMMC-7721/ADM by cis concentration gradient with initially concentration was IC50of SMMC-7721. Draw the growth curve and calculated the doubling time between SMMC-7721and SMMC-7721/ADM. Determined the IC50of Doxorubcin on SMMC-7721/ADM and resistance index by MTT. Western blotting was employed to detect the expression of MDR1in LO2,SMMC-7721and SMMC-7721/ADM.(2) miRNA microarray profiling was used to detect the differentially expressed miRNAs among LO2, SMMC-7721and SMMC-7721/ADM. RT-PCR was employed to detect the differentially miRNAs to verify the microarray results.Results(1)MTT analysis showed that the IC50of ADM on SMMC-7721was0.12±0.01μg/ml, Both cell lines had similar growth curve and doubling time was no significant difference(p>0.5). the IC50of ADM on SMMC-7721/ADM was2.96±0.12μg/ml, and resistance index was24.67. MDR1protein expression in SMMC-7721/ADM was higher than its parental cell SMMC-7721and LO2(p<0.05),also in the SMMC-7721was higher than LO2(p<0.05).(2) there are189miRNAs which up regulation more than2fold and246miRNAs which down regulation more than2fold between LO2and SMMC-7721,176miRNAs which up regulation more than2fold and159miRNAs which down regulation more than2fold between SMMC-7721and SMMC-7721/ADM,and160miRNAs which up regulation more than2fold and134miRNAs which down regulation more than2fold between LO2and SMMC-7721/ADM, was deteced by miRNAs microarray profiling. miR-1246, miR-3676-5p and miR-4443was in turn up regulation in LO2, SMMC-7721and SMMC-7721/ADM. miR-101-3p and miR-1469was in turn down regulation in LO2, SMMC-7721and SMMC-7721/ADM. RT-PCR analysis showed that the expression of miR-1246and miR-1469was consistent with miRNA array in LO2,SMMC-7721and SMMC-7721/ADM.Conclusion(1)Success to establish the drug resistance cell line SMMC-7721/ADM.(2)By miRNA microarray profiling, miR-1246, miR-3676-5p and miR-4443was in turn up regulation in LO2, SMMC-7721and SMMC-7721/ADM,miR-101-3p and miR-1469was in turn down regulation in LO2, SMMC-7721and SMMC-7721/ADM. The result of miRNA microarray was verified by RT-PCR. CHAPTER Ⅱ The study of miR-1246in drug resistance cell line SMMC-7721/ADMObjectiveTo explore the function of miR-1246in drug resistance cell line SMMC-7721/ADM.Methods(1) Design and synthesis miR-1246inhibitor fragment, and transfect to SMMC-7721/ADM, RT-PCR was used to determined the expression of miR-1246in SMMC-7721,SMMC-7721/ADM-inhibitor-NC,and SMMC-7721/ADM-ihibitor.(2)Experimental groups:A for SMMC-7721, B for SMMC-7721-inhibitor-NC, C for SMMC-7721-ihibitor.(3) MTT was used to determine the proliferation of such three cell lines and the IC50of Doxorubcin on SMMC-7721/ADM-ihibitor.(4) Detected the cell cycle of three cell lines by FCM.(5) Detected the cell apoptosis of three cell lines by FCM AV-PI.(6) Detected the migration ability of three cell lines by Transwell.(7) Detected the invasion ability of three cell lines by matrigel Transwell.Results(1)Transfection efficiency observed by microscope fluorescence was about85%, the expression of miR-1246in group C was lower than group B (p<0.05), and had no significant difference with group A(p>0.05).(2)MTT results showed that the proliferation of group C was lower than group A and B(p<0.05). there was no significant difference between group A and B(p>0.05). The IC50of ADM on group C was0.08±0.01μg/ml, and decreased significantly with the IC50of SMMC-7721/ADM(p<0.05). there was no significant difference in IC50between group C and A (p>0.05).(3) Cell cycle experiment illustrated that there was no significant difference among such three cell lines(p>0.05).(4)Apoptosis experiment showed that the group C has a higher apoptosis rate than group A and B(p<0.05). there was no significant difference between group A and B(p>0.05)(5)Transwell showed that the migration abitily of group C lower than group B(p<0.05), and had no significant difference with group A(p>0.05).(6) Invasion experiment showed that the invasion ability of group C lower than group B(p<0.05), and had no significant difference with group A(p>0.05).Conclusion(1)There is no significant difference between SMMC-7721/ADM and SMMC-7721in proliferation ability, cell apoptosis and cell cycle.but the ability of migration and invasion is improved. (2) In drug resistance cell SMMC-7721/ADM, miR-1246can increase the proliferation ability, inhibit cell apoptosis and had no effect on cell cycle.miR-1246can improve the ability of migration and invasion in SMMC-7721/ADM., Inhibit the expression of miR-1246would be sensitive to ADM on SMMC-7721/ADM. Chapter Ⅲ The bioinformatics analysis of miR-1246and the validation of target geneObjective(1) Predict the target gene via TargetScan, miRanDa and miRDB website. Select the intersection of three website as the target gene of miR-1246. GO annotation and KEGG biological pathway enrichment analysis was used to analyze those target genes via DAVID.(2) Selected GSK3β as target gene in SMMC-7721/ADM, and validated by western blotting and RT-PCR.Results(1)The number of intersection target gene was57,and KEGG showed that the pathway of those target gene was AXON GUIDANCE(P=4.8E-2).(2)The protein and mRNA of GSK3P were higher in SMMC-7721/ADM-inhibitor than SMMC-7721/ADM-NC(p<0.05), and had no significant difference with SMMC-7721.Conclusion(1)57candidate target gene of miR-1246was found.(2) GSK3β was one of the target gene of miR-1246in SMMC-7721/ADM.